Authors:Shivankar Agrawal; Alok Adholeya; Colin J. Barrow; Sunil Kumar DeshmukhPages: 5 - 13Abstract: Publication date: February 2018 Source:Anaerobe, Volume 49 Author(s): Shivankar Agrawal, Alok Adholeya, Colin J. Barrow, Sunil Kumar Deshmukh Cutibacterium acnes (or Propionibacterium acnes) is the main target for the prevention and medical treatment of acne vulgaris. The aim of this study was to evaluate the in vitro anti-C. acnes and anti-S. epidermidis properties of some marine fungi isolated from different Indian marine environments. Seventy fungal isolates were obtained from samples collected from the west coasts and Andaman Island, India. Methanol extracts of 35 isolates were screened for their antibacterial properties and 5 out of the 35 isolates displayed significant inhibition as compared with tetracycline. DNA was successfully extracted from these five fungal isolates and phylogenetic analysis was performed. The methanol extracts possessed antibacterial activity against C. acnes and S. epidermidis with MIC values ranged from 0.8 mg/mL to 1 mg/mL. SEM analysis revealed that the extract induces deleterious morphological changes in the bacterial cell membrane. This study has identified some fungi extracts with significant antibacterial activity. The extracts may have potential for development as an antibacterial agent in the treatment of acne vulgaris.

Authors:Xue Chen; Jumei Xu; Erdou Ren; Yong Su; Weiyun ZhuPages: 30 - 40Abstract: Publication date: February 2018 Source:Anaerobe, Volume 49 Author(s): Xue Chen, Jumei Xu, Erdou Ren, Yong Su, Weiyun Zhu The early development of gut microbiota plays a fundamental role in host health; so far, the main origins of the first colonization in newborn piglets are largely unclear. This study aimed to investigate the early development of gut microbiota in newborn piglets during lactation and their co-occurrence with microbes in the maternal and surrounding environments by Illumina MiSeq sequencing of 16S ribosomal RNA genes. The results showed that the microbial richness and diversity in piglets' feces (PF) significantly increased from birth to weaning (21 d). The composition and function of microbiota in the feces of piglets after birth tended to be similar to those from the slatted floor (FL), sow's milk (SM) and nipple surface (SN), and lacter, the fecal microbial communities of piglets later during lactation were more similar to their mother's. SourceTracker analysis showed that the microbiota from the FL, SM and SN were most likely the earliest passengers to the neonatal gastrointestinal tract, but did not have a long stay during lactation. The sow's fecal microbiota were easier to colonize in newborn piglet's guts via the co-occurrence effect with former settlers. This study suggests that microbes from the maternal and surrounding environments may play an important role in the microbial succession of newborn piglets after birth.

Authors:Anne Jolivet-Gougeon; Nicolas Helsens; Elise Renard; Zohreh Tamanai-Shacoori; Martine Bonnaure-MalletPages: 89 - 93Abstract: Publication date: December 2017 Source:Anaerobe, Volume 48 Author(s): Anne Jolivet-Gougeon, Nicolas Helsens, Elise Renard, Zohreh Tamanai-Shacoori, Martine Bonnaure-Mallet Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated for rapid identification of cfxA PCR positive and negative Capnocytophaga strains. Colonies were grown on blood agar, incubated anaerobically at 37 °C for 48 h, and were then evaluated by MALDI-TOF MS and 16S rRNA gene sequencing. Both methods identified all colonies to the genus level. The MALDI-TOF MS method gave the same result, at the species level, as 16S rRNA gene sequencing for 41/53 Capnocytophaga sp. strains (77.4%), but the limit of this technique was the absence of some species (C. leadbetteri, C. AHN) in the Biotyper-Bruker® database used in this study. Distinction between the cefotaxime resistant and susceptible strains was unsuccessful using the MALDI-TOF MS method. This technique had low discriminatory power to rapidly detect beta-lactamase-producing Capnocytophaga strains in clinical samples. However, the results from a score-oriented dendrogram confirmed MALDI-TOF MS is a rapid, inexpensive, and reliable method for Capnocytophaga species identification. Enrichment of the reference database used (Biotyper®) will improve future results.

Authors:A.C.M. Veloo; H. Jean-Pierre; U.S. Justesen; T. Morris; E. Urban; I. Wybo; H.N. Shah; A.W. Friedrich; T. Morris; H.N. Shah; H. Jean-Pierre; U.S. Justesen; E. Nagy; E. Urban; M. Kostrzewa; A. Veloo; A.W. FriedrichPages: 94 - 97Abstract: Publication date: December 2017 Source:Anaerobe, Volume 48 Author(s): A.C.M. Veloo, H. Jean-Pierre, U.S. Justesen, T. Morris, E. Urban, I. Wybo, H.N. Shah, A.W. Friedrich, T. Morris, H.N. Shah, H. Jean-Pierre, U.S. Justesen, E. Nagy, E. Urban, M. Kostrzewa, A. Veloo, A.W. Friedrich Inter-laboratory reproducibility of Matrix Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS) of anaerobic bacteria has not been shown before. Therefore, ten anonymized anaerobic strains were sent to seven participating laboratories, an initiative of the European Network for the Rapid Identification of Anaerobes (ENRIA). On arrival the strains were cultured and identified using MALDI-TOF MS. The spectra derived were compared with two different Biotyper MALDI-TOF MS databases, the db5627 and the db6903. The results obtained using the db5627 shows a reasonable variation between the different laboratories. However, when a more optimized database is used, the variation is less pronounced. In this study we show that an optimized database not only results in a higher number of strains which can be identified using MALDI-TOF MS, but also corrects for differences in performance between laboratories.

Authors:Károly Péter Sárvári; József Sóki; Miklós Iván; Cecília Miszti; Krisztina Latkóczy; Szilvia Zsóka Melegh; Edit UrbánPages: 98 - 102Abstract: Publication date: December 2017 Source:Anaerobe, Volume 48 Author(s): Károly Péter Sárvári, József Sóki, Miklós Iván, Cecília Miszti, Krisztina Latkóczy, Szilvia Zsóka Melegh, Edit Urbán Bacteroides fragilis as a commensal bacterium is a member of the human intestinal flora, but as an opportunistic pathogen it can cause serious infections as well. Some of them, harbouring an enterotoxin gene (bft), may cause diarrhoea mainly in young children. Recently it has been shown that a member of C11 proteases called fragipain (fpn) can activate the enterotoxin, while C10 protease (bfp) is suspected of playing an important role in the invasiveness of the B. fragilis isolates. The objective of this study was to investigate the prevalence and distribution of the bft isotypes in 200 Hungarian B. fragilis isolates collected recently; and in a subset of 72 strains, we wanted to determine the prevalence of bfp1-4 and fpn genes in bft-positive and bft-negative strains. Using the MALDI-TOF MS cfiA identification project file, 19 B. fragilis strains belonging to Division II were identified and the presence of the cfiA gene was confirmed by RT-PCR. Twenty six (13.0%) B. fragilis isolates turned out to be bft gene positive by RT-PCR; 20 isolates harboured bft-1 and six bft-2 isotypes, but no bft-3 isotype containing strains were found. A melting curve analysis and the PCR-RFLP were performed to differentiate between the bft-1 and bft-2 isotypes confirmed by sequencing. Thirty eight strains harboured bfp1, 58 isolates contained bfp2 gene, while 17 isolates proved positive for bfp3. Morever, no bfp4 positive isolate was found, and some of the B. fragilis strains tested harboured two or three bfp isotypes simultaneously. Among the 26 bft-positive strains, 24 contained the fpn gene, which confirms the role of fragipain in the activation of B. fragilis enterotoxin. In experiments, a significant negative correlation between fpn and cfiA was demonstrated (p < 0.000), a positive correlation was found between bfp2 and fpn genes (p = 0.0000803), and a negative correlation between bfp2 and cfiA genes (p = 0.011).

Authors:Zhi-Dong Jiang; Ashley Alexander; Shi Ke; Evangelia M. Valilis; Shaofan Hu; Bingjie Li; Herbert L. DuPontPages: 110 - 114Abstract: Publication date: December 2017 Source:Anaerobe, Volume 48 Author(s): Zhi-Dong Jiang, Ashley Alexander, Shi Ke, Evangelia M. Valilis, Shaofan Hu, Bingjie Li, Herbert L. DuPont Freezing donor fecal microbiota has simplified fecal microbiota transplantation (FMT) in the treatment of recurrent C. difficile infection (CDI). However, the optimal storage time for the frozen FMT products remains unknown. Using an established murine model of CDI, stability and efficacy of frozen and lyophilized FMT product was studied at time points from 2 months to 15 months. DNA was extracted from fecal samples from the mice with identification of specific bacterial species by real-time quantitative PCR (qPCR). FMT product stability and efficacy were measured by occurrence of diarrhea in the challenged mice together with stability of the microbiota composition. The results were analyzed and compared by SAS statistical software. All mice treated with only C. difficile developed diarrhea within 72 h. Mice treated with frozen (n = 5/group), lyophilized (n = 5/group) products stored for ≤ 7-month or fresh FMT product (n = 22) were protected from post C. difficile challenge diarrhea. There was no difference between frozen and lyophilized products (n = 5/group) stored for ≤ 7 months 95% CI 1.00 (0.38–2.64) and 1.00 (0.38–2.64), respectively. Prevention if CDI by frozen and lyophilized product was not different for storage of 9-, 11- and 15-months. qPCR results demonstrated there were no significant quantitative change in Bacteroides and Clostridium species during any of the storage times (P > 0.05). In the present study, frozen and lyophilized FMT products were stored up to 7 months without losing microbiota composition and therapeutic efficacy. The animal model described may be useful to study stability of human microbiota designed for FMT.

Authors:Luca Bano; Ilenia Drigo; Elena Tonon; Simone Pascoletti; Cinzia Puiatti; Fabrizio Anniballi; Bruna Auricchio; Florigio Lista; Cesare Montecucco; Fabrizio AgnolettiPages: 126 - 134Abstract: Publication date: December 2017 Source:Anaerobe, Volume 48 Author(s): Luca Bano, Ilenia Drigo, Elena Tonon, Simone Pascoletti, Cinzia Puiatti, Fabrizio Anniballi, Bruna Auricchio, Florigio Lista, Cesare Montecucco, Fabrizio Agnoletti Animal botulism is primarily due to botulinum neurotoxin (BoNT) types C, D or their chimeric variants C/D or D/C, produced by Clostridium botulinum group III, which appears to include the genetically indistinguishable Clostridium haemolyticum and Clostridium novyi. In the present study, we used matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI TOF MS) to identify and characterize 81 BoNT-producing Clostridia isolated in 47 episodes of animal botulism. The instrument's default database, containing no entries for Clostridium botulinum, permitted reliable identification of 26 strains at the genus level. Although supplementation of the database with reference strains enhanced the instrument's ability to identify the neurotoxic strains at the genus level, resolution was not sufficient to recognize field strains at species level. Characterization by MALDI TOF confirmed the well-documented phenotypic and genetic differences between Clostridium botulinum strains of serotypes normally implicated in human botulism (A, B, E, F) and other Clostridium species able to produce BoNTs type C and D. The chimeric and non-chimeric field strains grouped separately. In particular, very low similarity was found between two non-chimeric type C field strains isolated in the same outbreak and the other field strains. This difference was comparable with the differences among the various Clostridia species included in the study. Characterization by MALDI TOF confirmed that BoNT-producing Clostridia isolated from animals are closely related and indistinguishable at the species level from Clostridium haemolyticum and Clostridium novyi reference strains. On the contrary, there seem to be substantial differences among chimeric and some non-chimeric type C strains.

Authors:Sara Gómez; Fernando Chaves; M. Angeles OrellanaPages: 147 - 151Abstract: Publication date: December 2017 Source:Anaerobe, Volume 48 Author(s): Sara Gómez, Fernando Chaves, M. Angeles Orellana Recurrent diarrhea is a common complication of Clostridium difficile infection (CDI). Recurrent CDI (r-CDI) may be produced by the persistence of spores (relapse) or by the acquisition of a new strain (reinfection). In this study, we analyze epidemiological, clinical, microbiological and laboratory data from patients with r-CDI, relapse, and reinfection-CDI over 5 years and compared with a control group (non r-CDI). Among 60 patients with r-CDI, 36 patients had stool samples collected from two or more episodes, which were molecularly analyzed. Based on ribotyping, 63.9% of the samples were relapse, and 36.1% reinfection. In a multivariable logistic regression analysis, previous antibiotic exposure was found to be a risk factor for r-CDI (OR: 2.23; 95% CI: 1.0–4.9; p = 0.04). Patients with relapse had previous antibiotic exposure more frequently than did patients with reinfection (p = 0.03), and patients with reinfection suffered more frequently from chronic liver disease (p = 0.02) than did relapse patients. Relapse patients compared with the control group had a higher percentage of previous antibiotic exposure, although the difference was statistically no significant (73.9% vs. 91.3 p = 0.06). No significant differences for the selected variables were observed between the reinfection and control groups, although we observed a higher percentage of patients with chronic liver disease (30.8% vs 13.3%; p = 0.08). All isolates were sensitive to metronidazole and vancomycin. No significant differences in antibiotic susceptibility were found between the different groups. Sporulation and germination frequency of r-CDI were higher than non r-CDI (p = 0.02 and p < 0.01, respectively). Nevertheless, there were statistically not significant differences between the relapse and reinfection groups. Both frequencies were compared between the first and second episode of CDI for the relapse and reinfection groups, but differences were not observed to be statistically significant. In conclusion, our study showed that the recurrence of CDI was associated with antibiotic use and sporulation/germination frequency, regardless of relapse or reinfection. The use of antibiotics would produce a dysbiosis and favor the persistence of the C. difficile spores and relapse. A possible alteration of the intestinal microbiota and the bile salts produced by chronic liver disease could favor reinfection.

Authors:Yu Kajihara; Shota Yoshikawa; Yuichiro Cho; Toshiyuki Ito; Hirokuni Miyamoto; Hiroaki KodamaPages: 160 - 164Abstract: Publication date: December 2017 Source:Anaerobe, Volume 48 Author(s): Yu Kajihara, Shota Yoshikawa, Yuichiro Cho, Toshiyuki Ito, Hirokuni Miyamoto, Hiroaki Kodama Lactic acid produced by intestinal bacteria is fermented by lactate-utilizing bacteria. In this study, we developed a selective culture medium (KMI medium) for Megasphaera elsdenii, a lactate-utilizing bacterium that is abundant in pig intestines. Supplementation of the medium with lactate and beef extract powder was necessary for the preferential growth of M. elsdenii. In addition, we designed a species-specific primer set to detect M. elsdenii. When pig fecal samples were plated on KMI agar medium, approximately 60–100% of the resulting colonies tested positive using the M. elsdenii-specific PCR primers. In fact, nearly all of the large, yellow-white colonies that grew on the KMI agar medium tested positive by PCR with this primer set. The 16S rRNA gene sequences of three representative PCR-positive strains showed strong similarities to that of M. elsdenii ATCC 25940T (98.9–99.2% identity). These three strains were approximately 1.5 μm sized cocci that were primarily arranged in pairs, as was observed for M. elsdenii JCM 1772T. The selective KMI medium and species-specific primer set developed in this study are useful for the isolation and detection of M. elsdenii and will be useful in research aimed at increasing our understanding of intestinal short-chain fatty acid metabolism in pigs.

Authors:Ira Srivastava; Michael J. Aldape; Amy E. Bryant; Dennis L. StevensPages: 165 - 171Abstract: Publication date: December 2017 Source:Anaerobe, Volume 48 Author(s): Ira Srivastava, Michael J. Aldape, Amy E. Bryant, Dennis L. Stevens As the infectious disease paradigm undergoes a subtle shift, unusual infections associated with malignancy and immunosuppression are being increasingly reported. Spontaneous or non-traumatic Clostridium septicum infection is one such unusual infection which has gained prominence. This article aims to understand the pathophysiology, clinical manifestations and current trends in diagnosing and treating this rare but deadly infection. To understand the multifactorial causation of this infection a review of published cases of spontaneous C. septicum gas gangrene was performed and a total of 94 such cases were identified. Several factors were analyzed for each case: age, infection location and underlying illness, presenting signs and symptoms, neutropenia, gross pathology of the colon, antibiotic use, surgical intervention, and survival. A known or occult malignancy was present in 71% patients and an overall mortality of 67% was observed.

Authors:Jeanne Couturier; Catherine Eckert; Frédéric BarbutPages: 179 - 183Abstract: Publication date: December 2017 Source:Anaerobe, Volume 48 Author(s): Jeanne Couturier, Catherine Eckert, Frédéric Barbut MLVA analysis of 103 PCR ribotype 027 strains showed a regional specificity and the persistence of the same clone within a hospital several years apart. Capillary electrophoresis PCR ribotyping led to the identification of seven 027 variant strains and five 176 strains, four of them being implicated in an outbreak.

Authors:Song Zhang; Jieping Yang; Susanne M. Henning; Rupo Lee; Mark Hsu; Emma Grojean; Rita Pisegna; Austin Ly; David Heber; Zhaoping LiPages: 184 - 193Abstract: Publication date: December 2017 Source:Anaerobe, Volume 48 Author(s): Song Zhang, Jieping Yang, Susanne M. Henning, Rupo Lee, Mark Hsu, Emma Grojean, Rita Pisegna, Austin Ly, David Heber, Zhaoping Li Growing evidence suggests that dysbiosis of gut microbiota is associated with pathogenesis of a variety of human diseases. Using dietary intervention to shape the composition and metabolism of the gut microbiota is increasingly recognized. In the present study, we investigated the effects of polysaccharide inulin and polyphenol-rich pomegranate extract (PomX) alone or in combination on the cecal microbiota composition and function in a diet induced obesity mouse model. Male C57BL/6 mice were randomly divided into four experimental groups and consumed either high-fat/high-sucrose [HF/HS (32% energy from fat, 25% energy from sucrose, 17% energy from protein)] diet, HF/HS diet supplemented with PomX (0.25%), or inulin (9%) or PomX and inulin in combination for 4 weeks. In mice fed the PomX-diet the proportion of Turicibacteraceae and Ruminococcaceae was significantly increased compared to the control HF/HS diet. Supplementation with inulin alone and inulin + PomX combination significantly increased the proportion of Verrucomicrobiaceae (A. muciniphila) and decreased Clostridiaceae. Only mice fed the inulin diet experienced an increase in serum lipopolysaccharide (LPS) and monocyte chemoattractant protein 1 (MCP-1), which was reversed when feeding the inulin + PomX diet. Feeding the inulin + PomX diet was associated with a significant increase in Bifidobacteriaceae and Rikenellaceae, which may have contributed to the reduction of endotoxemia markers. Inulin supplementation showed lower species richness of gut microbiota compared to mice fed with HF/HS or HF/HS/PomX, and the reduction was reversed by the addition of PomX. Inulin alone and in combination with PomX had distinct microbial clusters determined by both weighted and unweighted UniFrac Beta-Diversity principle coordinate analysis. A total of 19 KEGG biological pathways were significantly regulated in the gut microbiota with PomX and inulin alone or combined treatment. Inulin significantly enhanced KEGG infectious disease-related pathway associated with increase of serum LPS and MCP-1. No changes in gene expression of ileal proinflammatory cytokine and tight junction genes were observed in mice treated with PomX and inulin. Our results demonstrated that the gut microbiota and their biological pathways were differentially effected by dietary PomX and inulin fed combined or alone. It is therefore very important to consider the interaction among bioactive components of food when evaluating potential prebiotic effects.

Authors:Jasmina Kerčmar; Albin PintarPages: 194 - 202Abstract: Publication date: December 2017 Source:Anaerobe, Volume 48 Author(s): Jasmina Kerčmar, Albin Pintar Hydrogen is considered to be an ideal energy alternative to replace environmentally burdensome fossil fuels. For its long-term production the immobilized biofilm system is the most promising and to choose the right support material the most challenging. In this respect, the anaerobic up-flow bioreactors packed with four most used support materials (polyethylene, polyurethane, activated carbon and expanded clay) were tested to investigate the crucial bacteria sensitive period-the immobilization process. Seven-day-operation was necessary and sufficient to reach metabolic and microbial stability regardless of support material used. The support material had an influence on the microbial metabolic activity as well as on quantity and quality characteristics of the immobilized microbial community, being polyethylene and expanded clay more appropriate as supports among the materials evaluated; this could be attributed to pH alteration. The obtained results suggest that the support material dictates the outcome of the immobilization process in the anaerobic continuous-flow bioreactor.

Authors:Qinqin Wu; Xiong'e Pi; Wei Liu; Huahai Chen; Yeshi Yin; Hongwei D. Yu; Xin Wang; Liying ZhuPages: 206 - 214Abstract: Publication date: December 2017 Source:Anaerobe, Volume 48 Author(s): Qinqin Wu, Xiong'e Pi, Wei Liu, Huahai Chen, Yeshi Yin, Hongwei D. Yu, Xin Wang, Liying Zhu Isomaltooligosaccharides (IMOs) are enzymatically synthesized oligosaccharides that have potential prebiotic effects. Five IMO substrates with 2–16° of polymerization (DP) were studied for their fermentation capacities using human microbiomes in an in vitro batch fermentation model. Eleven fecal slurries belonging to three enterotypes, including the Bacteroides-, Prevotella- and Mixed-type, exhibited different degradation rates for long chain IMOs (DP 7 to 16). In contrast, the degradation rates for short chain IMOs (DP 2 to 6) were not affected by enterotypes. Both 16S rRNA gene sequencing and quantitative PCR demonstrated that, after fermentation, the Bifidobacterium growth with IMOs was primarily detected in the Bacteroides- and Mixed-type (non-Prevotella-type), and to a lesser degree in the Prevotella-type. Interestingly, the Prevotella-type microbiome had higher levels of propionic acid and butyric acid production than non-Prevotella-type microbiome after IMOs fermentation. Moreover, principal coordinate analysis (PCoA) of both denaturing gradient gel electrophoresis (DGGE) profiling and 16S rRNA sequencing data demonstrated that the microbiome community compositions were separately clustered based on IMO chain length, suggesting significant impact of DP on the bacterial community structure. The current results clearly demonstrated that the IMO chain length could modulate the structure and composition of the human colonic microbiome. Different responses to short and long chain IMOs were observed from three human enterotypes, indicating that IMOs may be used as therapeutic substrates for directly altering human colonic bacteria.

Authors:I. Martín-Burriel; S. Andrés-Lasheras; F. Harders; R.C. Mainar-Jaime; B. Ranera; P. Zaragoza; V. Falceto; Y. Bolea; E. Kuijper; R. Bolea; A. Bossers; M. Chirino-TrejoPages: 224 - 231Abstract: Publication date: December 2017 Source:Anaerobe, Volume 48 Author(s): I. Martín-Burriel, S. Andrés-Lasheras, F. Harders, R.C. Mainar-Jaime, B. Ranera, P. Zaragoza, V. Falceto, Y. Bolea, E. Kuijper, R. Bolea, A. Bossers, M. Chirino-Trejo Clostridium difficile is an anaerobic spore-forming bacillus that usually causes gastrointestinal disorders in man and other animal species. Most of the strains isolated from animals are toxigenic being the virulent ribotype (RT) 078 predominant in several animal species. Although C. difficile is pathogenic to both humans and animals, there is no direct evidence of zoonosis. Deep genome sequencing provides sufficient resolution to analyse which strains found in animals might be related to human pathogens. So far, there are only a few fully sequenced genomes of C. difficile strains isolated from domestic and wild animals. Using Illumina technology, we have sequenced the genome of three isolates; a strain isolated from the vagina of a sow (5754), one from rat (Rattus spp) intestinal content (RC10) and a third one isolated from environmental rat faeces (RF17). Both, rat and rat faeces were sampled in fattening pig farms. Our study reveals a close genetic relationship of two of these isolates with the virulent strain M120 (RT078) isolated from a human patient. The analysis of the sequences has revealed the presence of antibiotic resistance genes, mobile elements, including the transposon linked with virulence Tn6164, and the similarity of virulence factors between these isolates and human strains. This is the first study focused on the sequencing of C. difficile genomes obtained from wild animals like rats, which can be considered as potential reservoirs for humans and other animal species. This study can help to understand the genome composition and epidemiology of this bacterium species.

Authors:Cecília Leite Costa; Cibele Barreto Mano de Carvalho; Rafael Holanda González; Markus Andret Cavalcante Gifoni; Ronaldo de Albuquerque Ribeiro; Carlos Quesada-Gómez; Gerly Anne de Castro BritoPages: 232 - 236Abstract: Publication date: December 2017 Source:Anaerobe, Volume 48 Author(s): Cecília Leite Costa, Cibele Barreto Mano de Carvalho, Rafael Holanda González, Markus Andret Cavalcante Gifoni, Ronaldo de Albuquerque Ribeiro, Carlos Quesada-Gómez, Gerly Anne de Castro Brito Clostridium difficile is a Gram-positive spore forming anaerobic bacterium and the main cause of healthcare-associated diarrhea. This study aimed to perform the phenotypic characterization and molecular typing of Clostridium difficile isolates among patients at a cancer hospital in Brazil. During 18 months, 48 diarrheic fecal samples were collected, of these 48% were positive in either one or both of the performed tests: detection of toxins A/B and culture. Clostridium difficile was recovered from four samples (17%). All strains carried toxin A and B genes, and the isolates belonged to PCR-ribotype 014/020, PGFE-type NAP4 and toxinotype XVIII. On the other hand, one isolate belonged to a novel PCR-ribotype, and PFGE-type, likewise to toxinotype IXb. The isolates showed susceptibility to metronidazole, vancomycin and moxifloxacin, and were resistant to ciprofloxacin. Finally, the findings indicate high positivity between the samples tested, suggesting an expressive importance of this infection, including detection of a novel ribotype/PFGE-type of Clostridium difficile, and show for the first time the detection of community-associated Clostridium difficile infection (CA-CDI) in these patients in Northeast Brazil. These data emphasize the importance to a better understanding of the epidemiological situation of this infection in Brazilian hospitals.

Authors:Patrizia Spigaglia; Fabrizio Barbanti; Elio Castagnola; Maria Cristina Diana; Luisa Pescetto; Roberto BandettiniPages: 262 - 268Abstract: Publication date: December 2017 Source:Anaerobe, Volume 48 Author(s): Patrizia Spigaglia, Fabrizio Barbanti, Elio Castagnola, Maria Cristina Diana, Luisa Pescetto, Roberto Bandettini Recent studies support a change of Clostridium difficile infections (CDIs) epidemiology in pediatric patients. Since limited information is available about C. difficile in this population, we investigated the epidemiology of CDI in a large pediatric hospital that acts as reference centre in Italy and analyzed C. difficile isolates to identify the prevalent PCR-ribotypes (RTs), the binary toxin (CDT)-positive strains and the antibiotic susceptibility patterns. The CDI incidence was 6.6 cases/1000 admissions and the majority (92%) of CDI were healthcare-associated (47% occurred in the Hematology-Oncology and in the Gastroenterology units). Most of symptomatic children <3 years with a positive culture for C. difficile were negative for other gastrointestinal pathogens, supporting C. difficile as cause of disease in these patients, including those showing recurrences. Strains RT020 (16%) and RT014 (14%) were identified as the main cause of infection, while RT356/607 and RT018, predominant in Italian adult patients, were absent (RT356/607) or rarely found (RT018) among children. CDT-positive strains represented the 20% of the total number of isolates analyzed. In particular, two emerging types, RT033 and RT442, were recognized as Toxin A-/Toxin B-/CDT+. Resistance to antibiotics characterized almost 50% of the toxigenic isolates analyzed in this study and, in particular, 20% of them were multidrug resistant (MDR). The emergence and circulation of strains with peculiar toxins profiles and/or MDR strongly highlight the necessity of a rapid CDI diagnosis, a careful monitoring of C. difficile in pediatric patients and a more strict control of antibiotics usage in the Italian pediatric hospitals.

Authors:Ritu Shrestha; Joseph A. SorgAbstract: Publication date: Available online 6 December 2017 Source:Anaerobe Author(s): Ritu Shrestha, Joseph A. Sorg Bile acids are an important signal for germination of Clostridioides difficile spores; however, the bile acid signal alone is not sufficient. Amino acids, such as glycine, are another signal necessary for germination by C. difficile spores. Prior studies on the amino acid signal required for germination have shown that there is a preference for the amino acid used as a signal for germination. Previously we found that d-alanine can function as a co-germinant for C. difficile spores at 37 °C but not at 25 °C. Here, we tested the ability of other amino acids to act as co-germinants with taurocholate (TA) at 37 °C and found that many amino acids previously categorized as non-co-germinants are co-germinants at 37 °C. Based on the EC50 values calculated for two different strains, we found that C. difficile spores recognize different amino acids with varying efficiencies. Using this data, we ranked the amino acids based on their effect on germination and found that in addition to d-alanine, other D-forms of amino acids are also used by C. difficile spores as co-germinants. Among the different types of amino acids, ones with branched chains such as valine, leucine, and isoleucine are the poorest co-germinants. However, glycine is still the most effective amino acid signal for both strains. Our results suggest that the yet-to-be-identified amino acid germinant receptor is highly promiscuous.

Authors:Karl-Jan Spittaels; Tom CoenyeAbstract: Publication date: Available online 22 November 2017 Source:Anaerobe Author(s): Karl-Jan Spittaels, Tom Coenye Aim The aim of the present study was to develop a new model system to study Propionibacterium acnes biofilms. This model should be representative for the conditions encountered in the pilosebaceous unit. Methods and results The new model, consists of an artificial sebum pellet supported by a silicone disc. Sebum pellets were inoculated with various P. acnes strains isolated from both normal and acneic skin. Growth and biofilm formation was verified by conventional plating at different time points, as well as by resazurin assays and fluorescence microscopy after LIVE/DEAD staining. The artificial sebum pellets were also used in assays to measure the production of certain virulence factors implicated in the pathogenesis of acne, including lipase, protease and the presence of CAMP factors. Conclusion The artificial sebum model can sustain biofilm growth of P. acnes, as was determined by increasing CFU counts for up to 1 week after inoculation. Metabolic activity and biofilm formation were confirmed using resazurin staining and fluorescence microscopy respectively. The production of virulence factors in this model was demonstrated as well.

Authors:Kazuko Okamoto-Shibayama; Jin Sekino; Kouki Yoshikawa; Atsushi Saito; Kazuyuki IshiharaAbstract: Publication date: Available online 13 October 2017 Source:Anaerobe Author(s): Kazuko Okamoto-Shibayama, Jin Sekino, Kouki Yoshikawa, Atsushi Saito, Kazuyuki Ishihara Treponemes occur in the microflora of the dental plaque. Certain Treponema species that are frequently isolated from chronic periodontitis lesions are involved in its initiation and progression. In addition to mechanical instrumentation, antimicrobial agents are used as an adjunctive treatment modality for periodontitis. Despite its importance for successful antimicrobial treatment, information about susceptibility is limited for Treponema species. The aim of this study was to assess the susceptibility of two Treponema denticola strains, Treponema socranskii, and Treponema vincentii to eleven antimicrobial agents. The minimum inhibitory and minimum bactericidal concentrations of these antimicrobial agents revealed strain-specific variation. Doxycycline, minocycline, azithromycin, and erythromycin were very effective against all Treponema species tested in this study, whereas fluoroquinolones only exhibited an equivalent effectiveness on T. socranskii. The susceptibility of one T. denticola strain, T. socranskii, and T. vincentii to kanamycin was influenced by prior exposure to aerobic conditions. The susceptibility to quinolone drugs varied among strains of T. denticola, although they share an amino acid sequence identity of greater than 99% for DNA gyrase (type II topoisomerase) subunit A. In addition, an ATP-binding cassette (ABC) transporter inhibitor assay for T. denticola indicated that the transport of quinolone drugs is partially related to this transporter, although there may be parallel transport mechanisms. Our results provide important insights into antimicrobial agent-Treponema dynamics and establish a basis for developing an appropriate adjunctive therapy for periodontal disease.

Authors:Yongrong Zhang; Zhiyong Yang; Si Gao; Therwa Hamza; Harris G. Yfantis; Michael Lipsky; Hanping FengAbstract: Publication date: Available online 12 October 2017 Source:Anaerobe Author(s): Yongrong Zhang, Zhiyong Yang, Si Gao, Therwa Hamza, Harris G. Yfantis, Michael Lipsky, Hanping Feng Most pathogenic Clostridium difficile produce two major exotoxins TcdA and TcdB, in the absence of which the bacterium is non-pathogenic. While it is important to investigate the role of each toxin in the pathogenesis of C. difficile infection (CDI) using isogenic strains, it is impossible to precisely control the expression levels of individual toxins and exclude bacterial factors that may contribute to the toxins' effects during infection. In this study, we utilized an acute intestinal disease model by injecting purified toxins directly into mouse cecum after a midline laparotomy. We evaluated the physical condition of mice by clinical score and survival, and the intestinal tissue damage and inflammation by histology. Depending on the dose of the toxins, mice developed mild to severe colitis, experienced diarrhea or rapidly died. We found that both purified TcdA and TcdB were able to induce clinical disease, intestinal inflammation, and tissue damage that resembled CDI. TcdA was significantly faster in inducing intestinal inflammation and tissue damage, and was approximately five times more potent than TcdB in terms of inducing severe gut disease and death outcomes in mice. Moreover, we found that the two toxins had significant synergistic effects on disease induction. Comparison of the in vivo toxicity of TcdB from clinical strains revealed that TcdB from an epidemic RT 027 strain was more toxic than the others. Our study thus demonstrates that both TcdA and TcdB, independent of other factors from C. difficile bacterium, are able to cause disease that resembles CDI and highlights the importance of targeting both toxins for vaccines and therapeutics against the disease.

Authors:Mirta R. Litterio; Daniela Cejas; Gabriel Gutkind; Marcela RadiceAbstract: Publication date: Available online 7 October 2017 Source:Anaerobe Author(s): Mirta R. Litterio, Daniela Cejas, Gabriel Gutkind, Marcela Radice CfiA (CcrA) metallo-β-lactamase is the main carbapenem resistance mechanism in B. fragilis. From cfiA positive isolates detected in a previous surveillance study, 3 displayed resistance to imipenem while the remaining were susceptible. The aim of this study was to identify the cfiA alleles and to analyze the presence of IS elements in their upstream regions. CfiA-1, CfiA-4, CfiA-13, CfiA-19 and CfiA-22 were detected. IS elements belonging to IS21 family and IS942 group were identified upstream to cfiA in the 3 imipenem resistant isolates. We present an exhaustive analysis of cfiA/CfiA registers in databases, illustrating the inconsistencies in both organization and nomenclature. According to this analysis CfiA family comprises nowadays 15 different CfiA variants coded by 24 cfiA sequences. Curation of CfiA database is mandatory, if not new cfiA admission at GenBank will contribute to make this classification more complex.

Authors:Reigadas Martin; BouzaAbstract: Publication date: Available online 6 October 2017 Source:Anaerobe Author(s): E. Reigadas, P. Muñoz-Pacheco, L. Alcalá, M. Marín, A. Martin, E. Bouza Background Rifaximin has been proposed as an alternative treatment for specific cases of Clostridium difficile infection (CDI) and intestinal decontamination. Rifaximin-resistant C. difficile has occasionally been reported. Antibiotic susceptibility testing relies on anaerobic agar dilution (reference method), which is cumbersome and not routinely used. There is no commercial test for detection of resistance to rifaximin. Objectives To assess resistance to rifaximin by C. difficile and to evaluate the correlation between the results of the rifampicin E-test and susceptibility to rifaximin. Methods We compared the in vitro susceptibility of clinical CDI isolates to rifaximin over a 6-month period using the agar dilution method with susceptibility to rifampicin using the E-test. All isolates were characterized using PCR-ribotyping. Clinical data were recorded prospectively. Results We recovered 276 consecutive C. difficile isolates and found that 32.2% of episodes were caused by rifaximin-resistant strains. The MICs for rifaximin ranged from <0.0009–256 mg/L, with a geometric mean (GM) of 0.256 mg/L, an MIC50/90 of 0.015/>256 mg/L. Rifaximin and rifampicin MICs were comparable, and all strains classed as resistant by agar dilution were correctly classified as resistant by E-test. The most common ribotypes were 001 (37.2%), 078/126 (14.3%), and 014 (12.0%). Ribotype 001 exhibited the highest MICs for rifaximin. Conclusions Resistance to rifaximin was common; resistance rates were higher in ribotype 001 strains. Susceptibility to rifaximin determined by agar dilution correlated with susceptibility to rifampicin determined using the E-test, including rifaximin-resistant strains. Our results suggest that the rifampicin E-test is a valid method for the prediction of rifaximin-resistant C. difficile.

Authors:R.S. Pereira; V.A.A. Rodrigues; W.T. Furtado; S. Gueiros; G.S. Pereira; M.J. Avila-CamposAbstract: Publication date: Available online 27 June 2017 Source:Anaerobe Author(s): R.S. Pereira, V.A.A. Rodrigues, W.T. Furtado, S. Gueiros, G.S. Pereira, M.J. Avila-Campos The quantification of ten microorganisms at the root ends and in the surrounding periradicular lesions was performed. Thirty 3 mm samples root ends and 30 samples of the surrounding chronic periapical infection were collected during apical microsurgery. Samples were triturated, and the bacterial DNA was obtained. The bacterial quantification was performed by using the SYBR Green system. At least one microorganism was detected in all patients. In both the root end and periapical samples, Fusobacterium nucleatum (71.6%), Dialister pneumosintes (58.3%) and Tannerella forsythia (48.3%) were the most prevalent species. Dialister pneumosintes showed statistically significant values in the root end, and F. nucleatum was also significant in the apical periodontitis samples. A statistically significant association between T. forsythia and Porphyromonas gingivalis in the root ends was observed. Bacterial associations from 2 to 7 species were observed in most samples. Extra-radicular and/or intra-radicular infections were present in all teeth with failed endodontic treatment, and showed polymicrobial infection in most cases, with a predominance of F. nucleatum, D. pneumosintes and T. forsythia. When present, Enterococcus faecalis was never found to be the most prevalent species. The presence of a microbial diversity in post-treatment apical periodontitis confirms the polymicrobial and synergistic characteristic of this process. Our results show that the bacterial array associated with the 3 mm root ends and periradicular lesions in post-treatment apical periodontitis are complex and with a high inter-individual variability. These results might be useful to delineate treatment strategies for microbial elimination in apical periodontitis. Further studies are necessary to elucidate the role of these microorganisms in endodontic treatment failures.

Authors:Rolf Claesson; Ulf Sjögren; Anders Esberg; Malin Brundin; Margareta GranlundAbstract: Publication date: Available online 21 June 2017 Source:Anaerobe Author(s): Rolf Claesson, Ulf Sjögren, Anders Esberg, Malin Brundin, Margareta Granlund There are few reports on the bacterial species Actinomyces radicidentis in the literature. In this study, putative A. radicidentis isolates were collected from 16 root canal samples from 601 examined patients. The isolates were examined by biochemical tests, 16S rRNA gene sequencing, Arbitrarily-primed (AP-) PCR, antibiotic susceptibility testing, and MALDI-TOF analyses. In parallel, two A. radicidentis reference strains and two putative A. radicidentis isolates from United Kingdom were tested. Sixteen of the 18 isolates were confirmed as A. radicidentis. The remaining two isolates, both of which were isolated from root canals (one from Sweden and the other from the UK), but were identified as Actinomyces haliotis by sequencing ∼ 1300 base pairs of the 16S rRNA-gene. This isolates had a divergent, but between them similar, AP-PCR pattern, and a common distribution of sequence signatures in the 16S rRNA gene, but were not identified by MALDI-TOF. A. haliotis is a close relative to A. radicidentis, hitherto only been described from a sea-snail. The identity of A. haliotis was confirmed by a phylogenetic tree based on 16S rRNA gene sequences with species specific sequences included, and by additional biochemical tests. The examined bacteria exhibited similar antibiotic susceptibility patterns when tested for 10 separate antibiotic classes with E-tests (bioMérieux). The MIC90 for β-lactams (benzylpenicillin and cefuroxime) and vancomycin was 0.5 mg/L, for colistin and ciprofloxacin 8 mg/mL and for the other antibiotic classes ≤ 25 mg/mL The isolation of A. haliotis from infected dental root canals cast doubt on the accepted opinion that all Actinomyces infections have an endogenous source.